We have identified a series of IGF1R-dependent human CRC cell lines that are highly metastatic to the liver and lungs after orthotopic implantation of the colon in athymic mice and are dependent upon IGF1R/EGFR crosstalk for cell survival. Survivin/XIAP complexes that mediate caspase inhibition have been shown to be a key cell survival mechanism for supporting the metastatic process. Recently, we identified a novel pathway in which cAMP-independent PKA activation by endogenous TGF? signaling resulted in the destabilization of survivin and XIAP thereby leading to their proteosomal degradation, caspase reactivation and cell death. Most importantly, the induction of this novel pathway prevented metastatic colonization of distant organs by highly metastatic IGF1R-dependent human cell lines following orthotopic implantation. Preliminary data now show that ligand-mediated activation of the IGF1R in these cell lines results in the generation of cAMP-dependent PKA activity. Therefore, this subset of CRC demonstrates two opposing pathways that both utilize activation of PKA that lead to different outcomes with respect to cellular life ad death. Understanding the specific mechanistic function of EGFR and IGF1R with respect to determining PKA survival functions would have potential for impact upon the development of combination treatment by these receptors. Therefore, Specific Aim I of the project will be directed at determining the individual functions of EGFR and IGF1R that promote cell survival and support metastatic potential. Preliminary data indicate that praja2, an AKAP-like ring ligase protein responsible for sustaining cAMP-dependent PKA activity through degradation of PKA regulatory subunits, is a key element in controlling cAMP-dependent PKA and pro-survival signaling through survivin/XIAP complex stabilization. Specific Aim II of the project will test the hypothesis that inhibition of praja2 function will result in disruption of metastatic capability an thus validate this understudied protein as a potential target for new treatment strategies for metastasis. We identified ezrin as a protein that was much more prevalent in metastases than primary tumors and, in addition, showed greater phosphorylation at T567 in metastases. This cytoskeleton-organizing protein has been implicated in the metastatic process of several other types of cancer in addition to CRC. Phosphorylation of ezrin at T567 locks ezrin into an open conformation thereby permitting its binding to a protein called EBP50 which is critical for the recruitment of ERM proteins that enable AKT signaling at the membrane. Ezrin T567 and EBP50 offer potential for novel anti-metastatic targets. Consequently, Specific Aim III will test the hypothesis that these two proteins are potential targets for anti-metastatic therapy. Since metastases are the major cause of death from solid tumors and there is currently little in the way of therapy for metastases that has a large impact on patient survival, the need for development of new therapeutic approaches is acute. PUBLIC HEALTH RELEVANCE: Metastases are the main cause of death from solid tumors as there are essentially no targeted therapies that have a large impact on survival when confronted with widely disseminated disease. This project undertakes the identification and genetic validation of novel targets in metastases to enable new therapeutic approaches against metastatic colorectal cancer.